ALBERT

Description: Alemtuzumab is a highly effective monoclonal antibody therapy for some B-cell disorders, and has been suggested as a possible therapeutic agent for treatment of myeloma. Monoclonal antibody therapeutic efficacy is closely associated with the expression level of the therapeutic target, as demonstrated by the lack of efficacy of single-agent rituximab in CLL. However, there are conflicting reports about the expression levels of CD52, the target for alemtuzumab, in plasma cell disorders. The aim of this study is to assess a large series of cases of plasma cell and B-cell disorders, utilising a standard approach to allow comparison of the target molecule. Plasma cells were assessed from patients with myeloma at presentation or relapse (n=106), monoclonal gammopathy of undetermined significance (MGUS, n=34), and from normal controls (n=19). In addition, B-cells were assessed from patients with chronic lymphocytic leukaemia (CLL, n=87), diffuse large B-cell lymphoma (DLBCL, n=10), follicular lymphoma (FCL, n=9), Waldenstroms macroglobulinaemia (WM, n=20), and also from normal bone marrow (n=37). Normal and neoplastic B-cells showed expression of CD52 (〉20% of cells above the CD3-control levels) in all patients except for 1/10 DLBL. B-CLL and WM are known to show responses to single-agent alemtuzumab therapy, and these two disorders had the highest levels of expression. In contrast, B-progenitor cells in normal bone marrow are unaffected by alemtuzumab, and proliferate during alemtuzumab treatment in CLL patients. The levels of CD52 expression by normal B-progenitors were 3-fold lower than CLL/WM. In DLBL and FCL, the B-cells showed very similar levels of CD52 expression to normal B-progenitors, on average 2.8-fold lower than CLL. All plasma cells, whether neoplastic (CD19− or CD19+56+) or normal (CD19+56−), showed much lower levels of expression than normal and neoplastic B-cells. Plasma cell CD52 expression was detectable in 68% of normal controls (13/19), 50% of MGUS patients (17/34), and only 43% of myeloma patients (46/106). Expression was uni-modal in all cases. There was significantly lower expression of CD52 by myeloma plasma cells than by their normal counterparts (median 2.4-fold decrease, P=0.03). Neoplastic plasma cell CD52 expression showed a high degree of inter-patient variation, but fewer than 10% of myeloma patients (7/106) had CD52 expression at a similar level to CLL cells. Neoplastic plasma cell CD52 expression was approximately 6-fold lower than that of normal B-progenitors, and nearly 20-fold lower than that of CLL cells. In summary, CD52 expression is not detectable above control levels in a significant proportion of myeloma patients. In cases with detectable CD52 expression, the antigen is at a much lower level than is present on normal B-progenitors, which actively proliferate during alemtuzumab therapy. The risk of immunosuppression due to depletion of residual normal B/T-cells must also be considered. As alemtuzumab efficacy appears to correlate with CD52 expression levels, myeloma is highly unlikely to respond to alemtuzumab as a single agent except in rare cases. However, alemtuzumab is more likely to be effective in the IgM immunosecretory disorders which show strong CD52 expression.

Description: B cell lymphomas with 100% Ki67 expression, represent a spectrum of disease incorporating Burkitt Lymphoma (BL). The currently recruiting MRC (UK) LY10 trial aims to determine the efficacy of CODOX-M/IVAC in adult BL. Due to the unfeasibility of demonstrating cMYC rearrangement prior to trial entry, 100% Ki67 is being used to define trial eligibility. The purpose of this study is to evaluate the heterogeneity of tumors selected on this basis, and to determine the effectiveness of 100% Ki67 as a surrogate marker for cMYC deregulation. To date we have immunophenotypic and FISH data on tumors from 145 patients. 93 cases have 100% Ki67 and are eligible for the trial. 62/93 (66.67%) cases were found to have a BL-specific phenotype (defined as CD20+, CD10+, BCL6+, BCL2−, P53+P21- and 100% cell cycle fraction) and of these 44 (71%) have rearrangement of cMYC. Rearrangement of cMYC was exclusive to cases with a BL phenotype, however 18/62 cases express the phenotype with no evidence of cMYC rearrangement. The remaining cases were GC- or activated-DLBCL. To test the hypothesis that cMYC may be deregulated by a mechanism other than gene rearrangement in cases with a BL-Phenotype, we have compared the gene expression profiles of samples expressing a BL-phenotype, but lacking a cMYC rearrangement (BL-Phen), with typical BL cases with rearranged cMYC (BLt), using Affymetrix Human U133 plus 2.0 chips. Due to the high levels of apoptosis in these tumors, cases with fresh material to extract sufficient RNA are rare, and only 8 cases were suitable for analysis. These were split into 3 groups: BLt (3); BL-Phen (3) and 2 DLBCL with 100% Ki67 but no other features of BL. A condition tree clustering algorithm was produced using GeneSpring v6.1 and correlation of the related samples was determined by Pearson correlation (P). In addition, a sub-analysis was performed on 33 genes reported to be downstream targets of cMYC. Using this approach, the 3 BLt cases clustered, as did the 3 BL-Phen cases, however comparison between groups showed a negative association (P= −0.3), suggesting that, although the phenotype is identical to BL, the molecular pathogenesis of BL-Phen cases does not involve deregulation of cMYC. A 〉3 fold increase in cMYC expression was found in the BLt compared to the BL-Phen group, and several genes, including nucleolin, ornithine decarboxylase 1, eukaryotic translation initiation factor 4E, carbamoyl-phosphate synthetase 2, high mobility group AT-hook 1, transferrin receptor and branched chain aminotransferase 1, were differentially expressed in BLt compared to BL-Phen cases. Interestingly, one of the non-BL-Phen samples had similar expression of cMYC and downstream genes as the BLt cases, suggesting cMYC activation by an alternative mechanism in this case. In conclusion, B-cell lymphomas with 100% Ki67 are a heterogeneous group of tumors, in which 47% are typical BL as defined by phenotype and cMYC rearrangement. Cases with a BL-Phenotype that lack cMYC rearrangement have an expression profile suggesting that cMYC is not deregulated, and that these patients may not be suitable for treatment with high intensity chemotherapy regimens. The data presented suggests that altered expression of the cMYC targets, as described, may be a more appropriate surrogate marker for rearrangement of cMYC, and it is likely that this approach will more accurately determine which patients will benefit from high dose therapy.

Description: Conventional therapy in CLL is not curative, partly because minimal residual disease (MRD) is usually detectable after therapy. Recent therapeutic approaches achieve higher complete remission (CR) rates, and often aim for MRD eradication as this has repeatedly been shown to predict improved outcome. However, many different MRD techniques have been used to assess response, making it extremely difficult to interpret and compare different clinical trials. The aim of this international collaboration is to develop standardized flow cytometric and PCR approaches to MRD monitoring in CLL that are broadly applicable; to assess their relative sensitivity and specificity in a variety of laboratories; and to establish a standardized reporting convention to facilitate the interpretation of clinical trials. These techniques may be used as a benchmark for assessing response and comparing the efficacy of different therapeutic approaches. PCR approaches using consensus primers to the immunoglobulin heavy chain (IgH) gene are straightforward and frequently used, but have highly variable sensitivity between patients. In the proposed standardized method, the IgH gene is amplified using the BIOMED-2 primers (van Dongen et al, Leukemia 2003, 17:2257) and an allele-specific CDR3 primer is designed. CLL cells are identified by RQ-PCR using the allele-specific oligonucleotide (ASO) primer coupled with intronic JH primers and consensus reporter probes. The amount of PCR product is compared to a single copy housekeeping gene (albumin) to identify the number of CLL cells in the sample. This approach reproducibly quantitates as few as 1 CLL cell in 10,000 other leucocytes, and may provide qualitative information below this level. Multi-parameter flow cytometric analysis with combinations such as CD19/CD5/CD79b/CD20 is reported to have a sensitivity approaching that of ASO-PCR. However, such approaches are less effective in situations where normal B-cells lack CD20, e.g. in patients treated with combinations including rituximab and in normal bone marrow. To circumvent these problems, the group has tested 58 individual four-colour combinations of antibodies identified from protein expression profiling and previously published studies. CD79b/CD43/CD19/CD5, CD81/CD22/CD19/CD5, and CD20/CD38/CD19/CD5 provided the least inter-laboratory variation, independent of methodology. They show low contamination by non-CLL cells and were therefore selected as a core panel for MRD analysis. Dilutional studies indicate that a quantitative sensitivity equivalent for detection of 1 CLL cell in 10,000 leucocytes is achievable; the practical sensitivity is being tested in blinded dilutional analyses. The proposed flow cytometry approach is applicable to all sample types and all therapeutic regimes, and sufficiently rapid and sensitive to guide therapy to an MRD negative status in real time. The PCR approach is highly sensitive but more complex, and is most suited to retrospective response assessment of clinical trials. We have identified the appropriate standardized analytical methods for both techniques and are currently validating these findings. These approaches will allow direct comparison of efficacy between different clinical trials, allowing more rapid international progress towards identifying a curative approach.